Figure 18.1 illustrates the steps in performing these tests. Each test involves centrifuging a citrated specimen of blood. Citrate complexes calcium, a necessary cofactor in the coagulation cascade, thereby preventing coagulation factor activation. The supernatant obtained by centrifugation, plasma, is incubated for 3 minutes with an additive that differs for the two tests. After addition of excess calcium with respect to citrate, the time to formation of gel is measured.

The PT, better called a complete thromboplastin time, incubates plasma with tissue extract (thromboplastin). As a tissue product, thromboplastin supplies its own phospholipid plus tissue factor. If the plasma sample contains sufficient factor VII and factors in the common pathway, it will gel in approximately 12 seconds. Variations in the potency and quality of thromboplastin reagents require simultaneous determination of a control sample and comparison of the patient’s result with the control result. Each batch of thromboplastin reagent is graded in potency. The international normalized ratio (INR) calculation allows for comparison of PT results from different commercial thromboplastin reagents: INR = PCR^ISI, where PCR is the ratio of patient sample to control sample PT results and ISI is the international sensitivity index, a measure of responsiveness to decreased concentrations of vitamin K-dependent factors. The higher the ISI of a thromboplastin, the less responsive the clotting time to changes in coagulation factors when using that thromboplastin. Therefore, a less responsive thromboplastin, which yields a smaller PCR, carries a larger ISI, yielding a similar calculated INR.
Thromboplastin reagent manufacturers determine the ISI by comparing the reactivity of each specific lot of thromboplastin with an international reference preparation (2).

The partial thromboplastin time (PTT) incubates plasma with an extract of thromboplastin that contains the phospholipid but not the tissue factor, thereby preventing activation of extrinsic factor VII. Now entirely dependent on surface activation alone, the gel forms slowly (73 ± 11 [SD] seconds) (3). The aPTT uses a surface activation accelerator such as kaolin, ellagic acid, silica, bentonite, or celite, which allows gel formation to then occur in approximately 32 seconds (1). As with the PT, simultaneous controls are required, with abnormal results at 1.5 or more times the control.

Warfarin therapy, which inhibits carboxylation of the vitamin K-dependent clotting factors, prolongs the PT because factor VII production is most vulnerable to lack of vitamin K. Heparin primarily affects the aPTT but not the PT because the potent procoagulant action of the thromboplastin reagent in the PT test easily overwhelms any inhibition of factors Xa and thrombin from the moderate doses of heparin used to treat venous thrombosis or acute coronary syndrome. The less potent partial thromboplastin reagent in the aPTT test, however, is sensitive enough to demonstrate heparin’s anticoagulant effects. Large doses of heparin will prolong the PT also.

Adding thrombin to a plasma sample will form fibrin within 10 seconds if functionally active fibrinogen is present, heparin is absent, and fibrin degradation products (FDPs) are absent. Sensitivity of the thrombin time to small amounts of heparin occurs because small amounts of thrombin are added. The traditional thrombin time is relatively insensitive to fibrinogen deficiency and to FDPs: detectable prolongation requires less than 0.75 g/L fibrinogen (4) or more than 200 mg/mL FDPs. Sensitivity to FDPs increases if fibrinogen concentrations are low. Also, diluted plasma increases sensitivity of the thrombin test to fibrinogen deficiency. Amyloidosis prolongs the thrombin time by inhibiting conversion of fibrinogen to fibrin (5).

The Reptilase time measures the interval between addition to plasma of venom from the South American pit viper Bothrops jararaca and formation of fibrin: it is like a thrombin time, but uses the venom instead of thrombin to form fibrin. A prolonged Reptilase time implicates reduced or dysfunctional fibrinogen or high concentrations of FDPs as the cause of a prolonged thrombin time. Neither heparin nor direct thrombin inhibitors prolong the Reptilase time (6). It is several-fold more insensitive to FDPs than the thrombin time. Amyloidosis also prolongs the Reptilase time.

Most laboratories use the Clauss method for fibrinogen determination, in which a thrombin time is performed on diluted plasma. With diluted samples, fibrinogen becomes the factor limiting clot formation, so that the clotting time varies inversely with fibrinogen activity (1). The Ellis method employs undiluted plasma, smaller amounts of thrombin, and a spectrophotometric measure of turbidity. A PT-based method adds thromboplastin to undiluted plasma, thereby using endogenously generated thrombin. Antibody-based tests for fibrinogen can distinguish among the dysfibrinogenemias. Decreased fibrinogen concentration occurs in end-stage hepatic failure, consumptive coagulopathy, and uncontrolled fibrinolysis leading to fibrinogenolysis, extreme hemodilution, and massive transfusion.

Immunologic tests provide a semiquantitative determination of the various fragments resulting from fibrin and fibrinogen degradation. The most common test for FDPs uses latex agglutination of serum. Note that serum, not plasma, is used. It provides results as either negative (<10 µg/mL) or positive (>10 µg/mL) for antigens, which are the breakdown products of either fibrin or fibrinogen. Serial dilutions of plasma yield more specific information when the undiluted sample is positive. More expensive quantitative analysis reveals normal serum levels of 2.1 to 2.7 µg/mL for FDPs in the absence of exercise or stress (1). This test does not differentiate the breakdown products of fibrinogen from those of fibrin.

Molecules of two linked “D” domains, shown in Figure 18.2, a specific degradation product of cross-linked fibrin, can be detected either semiquantitatively with a latex agglutination technique or in a fully quantitative manner with an enzyme-linked immunosorbent assay (ELISA). Many hospital coagulation laboratories offer this test on a batched basis. The D-dimer is more specific for secondary fibrinolysis than the FDP test. Like the FDP test, D-dimer results appear as fibrinogen equivalent units (i.e., the quantity of fibrinogen initially present that leads to the observed level of breakdown product). Normally, D-dimer is less than 0.5 µg/mL (fibrinogen equivalent units). Antibody-based tests use plasma, rather than serum, because the specificity afforded by the antibody method prevents fibrinogen in plasma from confounding the results.

The central role of platelets in coagulation and the impact of bypass on platelet function augment the importance of monitoring platelets during surgery. Because bypass affects both platelet function and count, measurement of platelet count is necessary but not sufficient to assess platelet role in coagulation. Although cell counters can and have been made mobile (7), measurement of platelet count has remained a central laboratory function at nearly all centers.

Platelet aggregometry utilizes a photo-optical instrument to measure light transmittance through a platelet-rich plasma sample (8). Upon exposure to a platelet agonist, the initially turbid sample shows increased light transmittance as platelets adhere to surfaces and one another. Impaired aggregation correlates poorly with clinical bleeding (8,9). Agonist agents include collagen, epinephrine, and adenosine diphosphate (ADP). Owing to the technical expertise often required to perform aggregometry, it finds application in research more often than in routine clinical care.

The injectable direct thrombin inhibitors hirudin, bivalirudin (Angiomax), and argatroban are alternatives to heparin available to patients with heparin-induced thrombocytopenia (HIT) (see Chapter 19). The ecarin clotting time (ECT) monitors the extent of anticoagulation from direct thrombin inhibitors. It is based on the conversion of thrombin to meizothrombin by ecarin, venom from the snake Echis carinatus (10). Less procoagulant than thrombin, meizothrombin binds to direct thrombin inhibitors in equimolar concentrations creating a linear correlation between ECT prolongation and thrombin inhibitor concentration (Fig. 18.3). With a point-of-care ECT test (11,12,13,14) no longer available, each hospital laboratory must create its own standardized version, thereby making the ECT extremely difficult to apply to the operative care of patients with HIT. Clinicians report successful cardiopulmonary bypass (CPB) using individual calibration curves for each patient (15). An alternative anticoagulation management scheme for patients with HIT takes the functional approach using either the activated clotting time (ACT) or aPTT,
as opposed to targeting specific blood concentrations using the ECT. Only case reports exist regarding monitoring failure or success (16,17,18,19,20,21).

Laboratory testing for heparin falls into two categories: clotting function and measurements of blood- or plasma heparin concentration. Advocates of clotting time cite the importance of assessing the clinical effect of heparin, suggesting that measuring concentration alone fails to detect patients resistant to anticoagulation effects of heparin (24). Proponents of concentration assays note the changing relation between ACT and blood heparin concentration induced by CPB, especially during hypothermia (25). Desirable characteristics of a heparin monitor include low cost, the use of whole blood, point-of-care availability with minimal equipment and operator attention, precise and accurate results that are quickly available, and the use of shelf-stable reagents (26).